Ogtr document

2.4 Crop Improvement

Australian canola has mainly been improved through recurrent selection in a closed population. This has led to inbreeding and genetic drift, with a loss of potentially valuable alleles (Cowling 2007). One of the major current challenges for Australian breeders is to introgress new genetic diversity, a key for adaptation to changing environments, while retaining the traits that were enhanced over the past 30 years. Germplasm from outside of Australia may provide valuable alleles for improvement. However, these imported germplasms need to be introgressed gradually, as they will most likely not be adapted to Australian conditions (Cowling 2007).

In 2006, the Australian Oilseed Federation (AOF) and the Grain Research and Development Corporation (GRDC) identified a series of agronomic and quality traits needed for canola germplasm development. They established the National Brassica Germplasm Improvement Program (NBGIP), defining five key priorities for improvement:

• 
  improved/alternative sources of blackleg resistance
  
• 
  increased water use efficiency/drought tolerance
  
• 
  reduced pod shatter
  
• 
  increased frost tolerance during seed development and
  
• 
  increased oil content stability and increased protein content (GRDC 2013; Salisbury et al. 2007).
  

Some more traits for germplasm enhancement, defined by NBGIP as preliminary and future traits are:

• 
  increased resistance to sclerotina, viruses and pests
  
• 
  improved early vigour
  
• 
  salt tolerance and
  
• 
  modified fatty acid composition for industrial uses (Amjad & Cowling 2007; Salisbury et al. 2007).
  

NBGIP proposes to use a set of methodologies, including marker assisted selection, interspecific hybridization, Target Induced Local Lesions in Genomes (TILLING) and incorporation of GM traits (Figure 4).

Figure 4: main strategies and tools available for canola improvement. Adapted from Redden et al. (2007) and Rahman (2013). QTL: Quantitative Trait Loci; SNP: Single Nucleotide Polymorphism; TILLING: Target Induced Local Lesions in Genomes. See text for details.

2.4.1 Breeding in Australia

Canola has moved in less than forty years from being a minor crop to one of the major oilseeds for food and feed industries in Australia and overseas (Wan et al. 2009). Australian public breeding programs started in 1970, in VIC, followed by NSW and WA (Buzza 2007; Salisbury & Wratten 1999). Private breeding began in 1980, a major focus being the development of hybrids (Salisbury & Wratten 1999). The first B. napus canola cultivars adapted to Australia growing conditions, Marnoo and Wesroona, were released in 1980 (Buzza 2007). The first canola-quality B. juncea variety for Australia, Dune, was released in 2007 (Burton et al. 2007).

See Potter et al (2016) and Salisbury et al (2016) for an extensive review and perspective of breeding progress in Australia since 1978.

Improved agronomic traits

Early canola varieties introduced into Australia from Canada were poorly adapted to the short days of the winter-spring growing season. One of the earliest aims of Australian breeders was to understand the flowering response and to delay the onset of flowering until after a satisfactory leaf canopy had developed (Walton et al. 1999) (Buzza 2007). Early and very early-maturing varieties, better adapted to drier environments, have been developed by breeding programs (Salisbury & Wratten 1999). The recent identification of Quantitative Trait Lociⁱ (QTL) involved in canola flowering response to photoperiod and temperature has been described as a promising avenue to adapt varieties to changing climates (Nelson et al. 2014). Raman et al (2016) recently reported the identification of QTL associated with yield and flowering time.

Breeders also recognised that growth and yield of canola would almost always be limited by water availability, particularly during seed set and maturation. Thus, improving water use efficiency and drought tolerance have been a major focus in canola breeding (GRDC 2007b; Wan et al. 2009). Because of its tolerance to drought and high temperatures, B. juncea has been used as an alternative to B. napus in low rainfall zones in a series of breeding programs (Oram et al. 1999).

Resistance to lodging and shattering are other sought-after traits (Hossain et al. 2012; Salisbury & Wratten 1999). Reduced plant height decreases the risk of lodging, while shattering resistance facilitates direct harvesting of canola (Salisbury & Wratten 1999). Improvements in these agronomic traits have increased yield, as considerable seed loss can occur due to lodging, shattering and the extra handling during windrowing.

Cleistogamy^j has also been described by some as a desirable agronomic trait in order to limit cross-pollination (Gruber et al. 2012). Cleistogamy does not exist naturally among the genetic resources of B. napus and B. juncea. However, lines of cleistogamous B. napus have been obtained using chemical induced mutagenesis strategies (Fargue et al. 2006; Leflon et al. 2010). The cleistogamous trait obtained has been described as imperfect: up to 72-89% of flowers were observed to be totally closed (Leflon et al. 2010). Pollen emission in cleistogamous plants was quantified as low as 10% of what is observed for open flowers (Fargue et al. 2006).

Resistance to blackleg

Blackleg disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of canola worldwide. In Australia, isolates of L. maculans have the ability to cause losses of up to 90% yield and it is predicted that, without management of the disease, the canola industry would disappear from Australia (Raman et al. 2012; Van de Wouw et al. 2014). The most severe epidemic observed in Australia occurred in 1972, causing a widespread collapse of the emerging canola industry (Buzza 2007; Li et al. 2007b). The varieties used were spring varieties from Canada, grown as winter crops and had not been selected for blackleg resistance (Buzza 2007). Since the late 1980s, Australian breeders have released a number of resistant lines, turning canola into a viable industry in the early 1990s (Li et al. 2007b). By the late-1990s, Australian mid-season varieties had the highest levels of blackleg resistance of any spring canola varieties in the world. These varieties were based on single dominant gene-derived resistance from B. rapa ssp. sylvestris (Li et al. 2007b). In 2003, the resistance was overcome, initially in WA and in other parts of southern Australia (Li et al. 2007b), threatening the industry. New sources of resistances are currently studied, using winter germplasm and polygenic resistance (Salisbury et al. 2007). The development of new resistances is to be associated with modified cropping practices, as detailed in the GRDC blackleg management guide (GRDC 2012). Another proposed strategy to minimise disease in crops is to use canola multilines^k cultivars or mixtures that have different resistance genes (Van de Wouw et al. 2014). Available lines with similar maturity time and herbicide resistance could be grown as a single crop, as this is done for wheat, barley and rice.

Non-GM herbicide tolerance

Canola is highly susceptible to weed competition during the early stages of growth, potentially leading to major yield losses. Excessive weed presence at harvest can also lower grain quality, thus potentially leading to more losses (GRDC 2009). Weed pressure from species, such as wild radish (Raphanus raphanistrum), wild turnip (Brassica tournefortii), Indian hedge mustard (Sisymbrium orientale) or Patterson’s curse (Echium plantagineum) was the main constraint to canola production in medium rainfall zones of southern Australia prior to the introduction of herbicide-tolerant varieties (Sutherland 2010).

The first non-GM herbicide-tolerant B. napus cultivar in Australia was a Triazine Tolerant (TT) canola, Siren, released in the mid-1990s. The first TT varieties released had a reduced radiation-use efficiency compared to non-TT lines, resulting in lower yields and lower oil content. Average yield penalty was about 15% (Pritchard 2014). This was compensated for by better weed control and TT varieties quickly captured the majority of the canola seed market. Current TT varieties have on average now closed the yield gap (Pritchard 2014). The first imidazolinone tolerant (IT, also known as Smart canola or Clearfield®) was released in Australia in 2000. IT varieties do not carry a yield penalty and have been widely adopted (Agriculture Victoria; accessed on 22 April 2016).

TT and IT canola varieties are both non-GM. The TT trait is derived from natural mutations observed in a wild biotype of B. rapa, transferred to B. napus through hybridization (Beversdorf & Kott 1987; Beversdorf et al. 1980). Tolerance is due to a single base pair change in the sequence of the chloroplast psbA gene encoding the D1 (QB) protein involved in electron transport of photosystem II (Reith & Straus 1987). IT was developed through chemical mutagenesis. The observed tolerance phenotype is due to mutations in the enzyme acetohydroxyacid synthase (AHAS), involved in the biosynthesis of branched-chain amino-acids (Swanson et al. 1989; Tan et al. 2005). IT varieties have been released for canola and also for corn (where the tolerance was first discovered), rice, wheat, sunflower and barley (Tan et al. 2005).

Fewer options are currently available for herbicide tolerance in commercial varieties of B. juncea. The first B. juncea canola IT varieties, OasisCL and SaharaCL, were released in 2008 (Potter et al. 2008). The first IT hybrid cultivar was released in 2013 (see Section 2.3.3 for more details). TT B. juncea varieties are currently being trialled in SA (EPARF 2015).

Non-GM herbicide tolerant varieties represent the vast majority of Australian B. napus canola-quality production, with 60% of TT cultivars and 15% IT (N. Goddard, personal communication, 2015). Very little detail is available regarding B. juncea. Details of currently available herbicide tolerant varieties can be obtained by consultation of various state government publications and the NVT website (accessed on 22 April 2016).

Improved oil and protein quality/quantity

As described above, one of the first aims of breeding in Australia was to produce canola-grade cultivars. Since then, the oleic acid content of mainstream Australian canola varieties has remained relatively constant at approximately 60%. However, further improvements and production of specialty varieties have been undertaken. One objective has been to further enhance oleic acid levels and reduce linolenic acid, to increase oil stability for specific applications such as deep-frying (Salisbury & Wratten 1999). HOLL (for High Oleic, Low Linolenic) B. napus cultivars have been developed, with up to 70% oleic acid content and less than 3.5% linolenic acid (Gororo 2007). Burton, 2009 suggested that B. juncea HOLL varieties could also be of interest for farmers. Other specialty cultivars for health products, such as omega-3 canola oil, are being developed, both in Australia and overseas, using conventional breeding and genetic modification (see below) (Potter et al. 2007).

Variety improvement has also focused on meal quality and digestibility, aiming at higher protein content and less fibre. These meals are low in glucosinolates, making them a suitable feed for poultry, pigs and cattle (AOF 2007).

Breeding has also focused on non-food, industrial applications. Specialty high erucic acid varieties have been developed, for use in the manufacture of paints, inks, nylon and plastic films (NSW Department of Primary Industries 2014). Canola-quality plants, particularly B. juncea canola could be used for biodiesel production (Haskins et al. 2009; McCaffery et al. 2009a). See Section 2.2 for more details.

Breeding and selection for oil with improved melting point, pour point and chemical stability has been proposed as a future target (NSW Department of Primary Industries 2014).

Hybrids as a breeding method

Overcoming genetic bottlenecks is critical for improvement of agronomic traits (such as shatter resistance or flowering time) but also for protecting the crop from diseases and pests (Osborn et al. 2007; Rahman 2013; Raman et al. 2014a; Redden et al. 2007). Intraspecific, interspecific and intergeneric crosses have been used by breeders to improve both oilseed and vegetable Brassica spp. crops. Hybrids are also widely used in breeding seeds for commercial planting due to heterosis^l, leading to increased yield performance and early vigour.

B. napus and B. juncea are largely self-pollinating (see Section 4.2 for more details) and the main constraint to commercial exploitation of hybrids has been the availability of an effective pollen control and fertility restoration system. The most efficient and widely used system is cytoplasmic male sterility (CMS). This system is based on genetic miscommunication between mitochondrial and nuclear genes, leading to abnormal anther and/or pollen development. There are three components to the system:

• 
  an A line carrying the mitochondrial genome leading to male sterility,
  
• 
  a B line, fully fertile, used to maintain the A line (A and B are sister lines^m),
  
• 
  a R line, with a nuclear gene restoring fertility. The R line should be highly heterotic to the A line.
  

The first non-GM B. napus hybrids based on a CMS system were released in Australia in 1988. These did not out-perform conventional varieties sufficiently to justify the higher seed cost. However, a number of hybrid B. napus varieties with improved yields have since become available to growers (McCaffery et al. 2006; Potter et al. 2007). CMS lines have also been developed for B. juncea, through wide hybridisation (Malik et al. 1999). Gene technology has also been used to develop hybrid production systems.

Interspecific and intergeneric crosses are an important source of gene diversity (Figure 4). Such crosses are often difficult, due to limited chromosome homology, abnormal meiosis or low recombination rates (Feng et al. 2009; Navabi et al. 2011). Several techniques have been developed to increase the breadth of germplasms available for crossing. Embryo rescue is routinely used to overcome species barriers in difficult crosses. Immature embryos are excised from the ovaries and grown on artificial media, to avoid abortion by the plant (Navabi et al. 2011). To avoid sexual incompatibility barriers, nuclear and cytoplasmic genomes can also be combined by protoplast fusion (also known as somatic hybridization). Protoplast fusion has been used to alter fatty acid composition in seeds or increase resistance to blackleg (Hu et al. 2002). So far, most somatic hybrids have shown a high degree of sterility and/or have exhibited morphological abnormalities. These hybrids are mostly used as bridges to transfer specific desirable traits.

Haploids and doubled haploids are also used to generate hybrids. Haploid cells from pollen or egg cells are isolated and cultured in vitro and chromosome doubling is chemically induced (often using colchicine). Doubled haploid lines are used more often than haploid ones, for they are more stable and fertile. Doubled haploids are completely homozygous and can be used in interspecific crosses, especially when these crosses involve parents with different levels of ploidy (Mason et al. 2015; Rahman 2013). Doubled haploids have been considered as an option to create new hexaploid species (Mason et al. 2015). Natural polyploidy in Brassica is confined to the occurrence of tetraploid plants. There are no hexaploid or higher polyploid Brassica species. Combining the three A, B and C genomes could produce varieties with increased tolerance to abiotic stresses such as drought or salinity and diseases (Pradhan et al. 2007; Pradhan et al. 2010). So far, breeding of hexaploid lines has been limited by high chromosomal instability and infertility (Chen et al. 2011).

Use of molecular techniques in breeding

Marker assisted selection and chromosome mapping started in the 1980s for canola, with the development of RFLP, AFLP and other genetic markers. These markers were used to produce the first linkage maps for B. rapa and B. napus in the early 1990s (OECD 2012). Other, more powerful genetic tools have since been developed, leading to the construction of high-resolution genetic maps.

Genetic markers such as RFLP, AFLP or SSR are used routinely to identify QTL. These identified QTL can then be used for breeding, to improve agronomic qualities such as flowering time and photoperiod responsiveness (Nelson et al. 2014), concentration of glucosinolates (Harper et al. 2012) or resistance to diseases (Hayward et al. 2012). Two high density QTL maps have recently been constructed for B. juncea, using crosses of eastern European and Indian varieties. These maps showed that yield-related QTLs in B. juncea were originating from the A genome rather than from the B genome (Ramchiary et al. 2007; Yadava et al. 2012).

Complete, annotated reference genome sequences for B. rapa (Wang et al. 2011b), B. napus (Chalhoub et al. 2014) and B. oleracea (Liu et al. 2014) are now publicly available. Such tools are predicted to help gene discovery and breeding of Brassicas (Wang & Freeling 2013). Computational methods have been used to analyse the structure of the B. rapa genome and compare it with Arabidopsis (Tang & Lyons 2012).

Recent advances in molecular techniques, such as Next Generation Sequencing (NGS), have made the characterisation of candidate resistance genes easier. By using whole-genome shotgun reads of the parents of a population segregating for resistance to blackleg, it has been possible to identify two candidate genes in a major resistance locus, Rlm4 (Tollenaere et al. 2012).

NGS has led to Single Nucleotide Polymorphism (SNP) being widely used for QTL mapping and comparative genomics. In particular, deep transcriptome RNA sequencing (RNA seq) has reduced costs as SNP detection can focus on coding regions only (Devisetty et al. 2014a). B. napus, B. juncea and B. rapa genomes have recently been investigated using SNP-based fine mapping methods (Devisetty et al. 2014b; Raman et al. 2014a). Distribution and frequency of SNP are important data for their use as genetic markers. SNP rate among B. rapa cultivars is of about 1 in 150-200bp, while it is of about 1 in 1.6kb between two cultivars of B. napus (Devisetty et al. 2014a). SNP frequency observed in Brassica spp. is within the range of those reported for other plant species.

TILLING is a direct, cost-efficient reverse genetics technique for point mutation or SNP screening. It is used in natural or mutagenized populations (following treatment with a chemical mutagen such as ethyl methanesulfonate). Combining TILLING and NGS helps identifying mutants in polyploid species and will be of interest for breeders (Gilchrist et al. 2013).

2.4.2 Genetic modification

Genetic transformation of canola started in the late 1980s and early 1990s, with the first commercial release in 1994 in the US. Both biolistics and Agrobacterium tumefasciens-based nuclear transformation techniques are used routinely, with methods used for Arabidopsis adapted for B. napus and then B. juncea (Chhikara et al. 2012; Dutta et al. 2008; Wang et al. 2003). Hypocotyls, cotyledons, stems, leaf discs, microspores or protoplasts can be used to regenerate GM plants (see (Dutta et al. 2008) for details). As for Arabidopsis, Agrobacterium-mediated transformation of B. napus and B. juncea can be done by floral dip, by vacuum-infiltrating immature floral buds (Chhikara et al. 2012; Wang et al. 2003). Floral dip transformation efficiency is quite low: about 0.8% of seeds analysed by Chhikara et al. (2012) were found positive by Southern blot. Floral dip is routinely used as no tissue culture is required, thus reducing time and cost associated with transformation.

A protocol for chloroplast transformation of B. napus has been described recently (Cheng et al. 2010). Chloroplast transformation offers several advantages compared to nuclear transformation. The method is based on homologous recombination, making it a high-precision engineering technique. Chloroplasts are prokaryotic and multiple transgenes can be stacked, if linked together as operons. Furthermore, there is no epigenetic control or gene silencing mechanisms in chloroplasts. Thus the risk of transgene non-expression is reduced compared to nuclear transformation (Clarke & Daniell 2011).

GM canola varieties commercially released so far worldwide have been genetically modified for herbicide tolerance, high oleic acid content and/or a hybrid breeding system. Current laboratory work and field work in Australia and overseas mainly focus on pathogen resistance (Zhang et al. 2015), abiotic stress tolerance (Chakraborty et al. 2012), oil quality (Tan et al. 2011) or yield (Kant et al. 2015).